Evelyne Bertrand

Box C/D small nucleolar RNA trafficking involves small nucleolar RNP proteins, nucleolar factors and a novel nuclear domain

Nucleolar localization of box C/D small nucleolar (sno) RNAs requires the box C/D motif and, in vertebrates, involves transit through Cajal bodies (CB). We report that in yeast, overexpression of a box C/D reporter leads to a block in the localization pathway with snoRNA accumulation in a specific sub‐nucleolar structure, the nucleolar body (NB). The human survival of motor neuron protein (SMN), a marker of gems/CB, specifically localizes to the NB when expressed in yeast, supporting similarities between these structures. Box C/D snoRNA accumulation in the NB was decreased by mutation of Srp40 and increased by mutation of Nsr1p, two related nucleolar proteins that are homologous to human Nopp140 and nucleolin, respectively. Box C/D snoRNAs also failed to accumulate in the NB, and became delocalized to the nucleoplasm, upon depletion of any of the core snoRNP proteins, Nop1p/fibrillarin, Snu13p, Nop56p and Nop5p/Nop58p. We conclude that snoRNP assembly occurs either in the nucleoplasm, or during transit of snoRNAs through the NB, followed by routing of the complete snoRNP to functional sites of ribosome synthesis.

The long-acting gonadotropin-releasing hormone analogues impaired the implantation rate.

OBJECTIVES To determine the efficacy and innocuousness of long-acting versus short-acting GnRH analogues (GnRH-a) in long protocol for in IVF-ET. DESIGN Prospective randomized study. SETTING The IVF unit at an academic hospital. PATIENTS One hundred couples admitted for their first IVF-ET attempt. MAIN OUTCOME MEASURES Serum concentrations of LH, E2, and P during the all cycles and duration of pituitary desensitization were assessed, as well as fertilization rate, embryo quality, and implantation and pregnancy rates. RESULTS Significantly more days (10.8 +/- 1.8 versus 9.2 +/- 1.7 days) of stimulation and more ampules of hMG (47 +/- 22 versus 33 +/- 16) were necessary to obtain similar numbers of embryos of quality with the long-acting GnRH-a. Implantation and delivery rates were significantly lower with the long-acting GnRH-a (32.8% versus 21.1%; 48.9% versus 29.1%, respectively). CONCLUSIONS As the long-acting GnRH-a might interfere with the luteal phase and embryo development, short-acting GnRH-a should be preferred for ovarian hyperstimulation in IVF-ET.

Presence of HIV-1 in follicular fluids, flushes and cumulus oophorus cells of HIV-1-seropositive women during assisted-reproduction technology.

HIV-1 RNA and DNA were measured in follicular fluids, flushes and cumulus cells during eight cycles of in-vitro fertilization/intracytoplasmic sperm injection in four infected patients. No production of HIV-1 RNA or DNA was evidenced in the follicular fluids or cumulus cells of patients with undetectable plasma viral loads. In the one patient with a detectable plasma viral load, HIV-1 RNA was detected in a sixth of the samples tested. Her baby remained HIV-1 negative.

Intracytoplasmic single sperm injection: preclinical training and first clinical results

ObjectiveOur purpose was to reduce oocyte damage before clinical application of intracytoplasmic single sperm injection by training on aged unfertilized oocytes.DesignIntracytoplasmic single sperm injection (ICSI) was accomplished by micromanipulation of sperm and oocytes.PatientsThirty-four patients consented to donate unfertilized aged oocytes to train for ICSI. Forty-four patients suffering from severe male infertility were treated with ICSI.InterventionOocytes were inseminated by intracytoplasmic single sperm injection.Main outcome measuresOocyte damage and fertilization and pregnancy rates were the outcome measures.ResultsOne hundred fifty-one aged unfertilized oocytes were gathered for training of which 121 were injected with a single sperm and 30 without a spermatozoon as a control group for activation. Oocyte damage, initially as high as 40%, was reduced to 15% after 60 oocytes. Normal fertilization (2PN) occurred in 18%, and polyploidy in 4.4%. The cleavage rate was 69%; none of these embryos were transferred. In the control group, seven oocytes were damaged, seven (30%) showed one pronucleus, and one showed two pronuclei. No cleavage was observed in the control group. In the clinical trial, 44 patients (61 cycles) were clinically treated with the same ICSI procedure, including 575 of 721 collected oocytes. Damage was 13%, activation was 11%, normal fertilization was 30%, and 5 (1%) polypoid zygotes were observed. The fertilization rate ranged from 5 to 100%, with a mean of 39.5±4% (SE). Nine patients had no fertilization (15%). Ninetysix percent of the zygotes cleaved and 47% were at the four-cell stage 45 hr after injection. One hundred twelve embryos were replaced in 48 transfers (2.3 embryos/ET). One live birth, one miscarriage, and eight ongoing pregnancies were obtained (22%/ET).ConclusionPreclinical practice on aged unfertilized oocytes seems useful before starting clinical ICSI, as the high initial oocyte damage can be reduced and subsequent clinical treatment successfully applied.

Abnormal sperm-mucus penetration test predicts low in vitro fertilization ability of apparently normal semen

OBJECTIVE To investigate whether Kremers sperm-mucus penetration test may predict sperm fertilizing ability in IVF. DESIGN Kremers test was prospectively performed on semen samples used for 66 consecutive IVF trials and compared with the fertilization rates and fertilization failure rates observed. RESULTS Fertilization rates were significantly reduced in cases of abnormal Kremers test (42% versus 51%; n = 745 oocytes with a statistically insignificant increase in fertilization failure rates (21% versus 10%; n = 66 trials). For abnormal semen, fertilization rates (39% versus 39%; n = 208 oocytes) and fertilization failure rates (20% versus 28%; n = 17 trials) were similar regardless of Kremers test result. For normal semen, an abnormal Kremers test implied a significant decrease in fertilization rates (44% versus 54%; n = 537 oocytes) with a statistically insignificant increase in fertilization failure rates (21% versus 6%; n = 49 trials). CONCLUSIONS Abnormal Kremers test results identify patients with a decreased in vitro fertilizing ability despite apparently normal semen samples and a group with very low fertilizing failure risk in case of normal semen samples and normal Kremers test. Kremers test does not add any predictive value to sperm analysis in the case of abnormal semen samples. These observations point out the importance of the male factor in fertilization failure even in the case of normal semen analysis.

Quality control in IVF with mouse bioassays : A four years' experience

AbstractObjective: We analyzed retrospectively the relevance of 4 years of quality control on homemade culture medium with the mouse IVF and zygote bioassay. Design: In vitro or in vivo fertilized mouse oocytes were cultured in each batch of medium. Two-cell-stage and expanded blastocyst development was recorded for each batch of medium. Data on fertilization and embryo quality obtained in human in vitro fertilization were recorded for each batch. IVF treatment cycles for male infertility and cycles with sperm microinjection were excluded. Results: Human oocyte fertilization dropped from 60 to 54%, respectively, from 57 to 41% in a significant way (P<0.05 resp. P<0.01) and the human mean embryo score decreased from 4.17±1.21 to 3.69±1.06 (P<0.05) when media were used with a low two-cell-stage development (≤75%) for the mouse zygote or mouse IVF bioassay. The pregnancy rate was not affected. Conclusions: Media with high scores in mouse bioassays show higher fertilization rates and better embryo quality when used for human IVF.

Second polar body extrusion is highly predictive for oocyte fertilization as soon as 3 hr after intracytoplasmic sperm injection (ICSI)

ObjectiveOur objective was to evaluate the time course and the predictive value of the extrusion of the second polar body after intracytoplasmic injection (ICSI) related to the fertilization rate, embryo cleavage and quality.SettingThe setting was the in vitro fertilization program of a university hospital.PatientsTwenty-one patients were treated with intracytoplasmic single sperm injection either for fertilization failure in IVF, low fertilization in IVF (<5%), or severe male factors.DesignOne hundred thirty-five of 205 metaphase 2 oocytes treated with intracytoplasmic single sperm injection were observed 1, 2, and 3 hr after the assisted fertilization procedure. Extrusion of the second polar body was recorded. For each of these oocytes, fertilization was noted 18 hr after ICSI and cleavage and embryo quality were assessed 24 hr later. The 70 remaining oocytes were used to assess a possible negative effect of repeated exposure to light microscopy.ResultsThe extrusion of the second polar body 3 hr after injection was an observation with a sensitivity of 0.87, a specificity of 0.58, and a high positive predictive value (0.90) toward oocyte fertilization. Twenty-nine and four-tenths percent of the oocytes extruded a second polar body within the first hour, 56.6% within the first 2 hr, and 78.3% had a second polar body 3 hr after injection. This time course was related neither to the speed of embryo cleavage nor to the embryo quality. Fertilization, cleavage, and embryo quality were not affected by repeated observation as deduced from comparison with the control group and confirmed by a high pregnancy (62% per oocyte retrieval) and implantation rate (22% per replaced embryo).ConclusionOocytes can be checked, in all safety, 3 hr after a single sperm injection for the presence of a second polar to predict oocyte fertilization with a high certainty.

Oocytes affected by smooth endoplasmic reticulum aggregates: to discard or not to discard?

PurposeOocytes containing smooth endoplasmic reticulum aggregates (SERa) have been associated with reduced fertilization and clinical pregnancy rates as well as compromised neonatal outcomes. It was therefore recommended by an Alpha-ESHRE Consensus to discard oocytes presenting this dysmorphism. The data in the literature are nevertheless conflicting and healthy babies have recently been obtained from affected oocytes. The objectives of this study were to compare clinical outcomes between ICSI cycles with and without oocytes affected by smooth endoplasmic reticulum aggregates and to confirm whether affected oocytes can produce healthy babies.MethodsA prospective observational study was performed comparing 714 SERa− ICSI cycles to 112 SERa+ cycles. Among the SERa+ cycles, 518 SERa− oocytes and 213 SERa+ oocytes were analyzed. Fertilization, embryo quality, and pregnancy rates as well as neonatal outcomes were compared between SERa+ and SERa− cycles as well as between SERa+ and SERa− oocytes.ResultsThe presence of SERa was not associated with an adverse effect on embryological, clinical or neonatal data for SERa+ cycles and oocytes. Seven healthy babies were born from embryos originating from SERa+ oocytes.ConclusionsThese results are encouraging and might contribute in the future to a revision of the Alpha-ESHRE Consensus. Larger studies, including a correlation between frequency and size of SERa, clinical outcomes and malformation rates, as well as the follow-up of babies born are nevertheless necessary. In the meantime, the currently conflicting data requires caution when considering transfers of embryos affected by SERa.

Clinical parameters influencing human zona pellucida thickness**Supported by the Fonds National de le Recherche Scientifique, Brussels, Belgium.

OBJECTIVES To assess which clinical parameters influence human oocyte zona pellucida (ZP) thickness. PATIENTS Sixty-five couples undergoing 75 IVF-ET cycles. MAIN OUTCOME MEASURE Zona pellucida thickness of 827 oocytes measured 16 to 20 hours after in vitro insemination under inverted light microscope. RESULTS Zona pellucida thickness was 18.9 +/- 3.8 microns (mean +/- SD) for unfertilized, 16.4 +/- 3.1 microns for fertilized, and 15.1 +/- 2.4 microns for polyspermic oocytes (significantly different). Among our patients, a few underwent two (or even three) IVF-ET cycles, and the mean ZP thickness was, in most cases, not significantly different from one cycle to the other(s). Regression analyses were calculated between ZP thickness and available clinical parameters, i.e., the age of the women, the duration of stimulation, the cumulus maturity, the number of retrieved oocytes, the number of hMG doses, the maximum E2 level, and the follicular volume. A significant linear decreasing relationship exists between the mean ZP thickness of each patient and the maximum E2 level and an increasing one with the hMG dose. Relationships with the other parameters appeared to be nonsignificant. CONCLUSION The ZP thickness is basically an individual feature that influences the fertilization rate. Nevertheless, it may be influenced slightly by the hormonal treatment during stimulation.

Physiologie de l’interaction gametique: Revue de la litterature

Our knowledge of gamete interaction in vitro and in vivo has grown rapidly over the last century. Its importance has been reinforced by the development of in vitro fertilization and assisted conception procedures on one hand, and by research on the development of contraceptive vaccines on the other. This literature review synthesizes our current understanding of the successive steps in the fertilization process from sperm capacitation allthrough gamete fusion, including sperm interaction with the cumulus oophorus and the zona pellucida, as well as consideration on the mechanisms and timing of the acrosome reaction.ResumeL’interaction gamétique, in vitro comme in vivo, est un domaine ou l’acquisition des connaissances a été très rapide au cours de ce siècle. Son importance s’est encore renforcée avec le développement de la fécondation in vitro et la fécondation assistée d’une part, et des recherches de vaccins contraceptifs d’autre part. La revue de la littérature présentée ici fait le point sur les différentes étapes de ce phénomène, de la capacitation du spermatozoïde à la fusion gamétique en passant par l’interaction du spermatozoïde avec le cumulus oophurus et la zone pellucide et par les mécanismes et la chronologie de la réaction acrosomique.